1. Field of the Invention
The present invention relates to a modified reductase that can be used for reduction reaction, specifically reduction reaction of β-keto acid, and the like, and its gene and use thereof.
2. Background of the Invention
Reductases have been used recently for an organic synthesis reaction for production of compounds used as active ingredients of medicaments or agrochemicals or intermediates thereof, especially optically active compounds or intermediates thereof.
According to the present invention, there is provided a modified reductase having a wild-type amino acid sequence in which a certain amino acid has been substituted, which can produce reaction products with good optical purity.
The present invention provides:
(1) a reductase comprising
(i) an amino acid sequence of SEQ ID NO:1 having a substitution at amino acid position 54 or 104 or at both of the amino acid positions 54 and 104, or
(ii) an amino acid sequence defined in (i) having further deletion, substitution, or addition of an amino acid or acids, (hereinafter referred to as a reductase of the present invention);
(2) a reductase according to (1) above, which comprises
an amino acid sequence of SEQ ID NO:1 having
a substitution at amino acid position 54 or 104 or at both of the amino acid positions 54 and 104, and
further substitution of an amino acid or acids;
(3) a reductase according to (1) above, wherein said substitution is a single amino acid substitution at amino acid position 54;
(4) a reductase according to (1) above, wherein said substitution is a single amino acid substitution at amino acid position 104;
(5) a reductase according to (1) above, wherein amino acids at amino acid positions 54 and 104 are substituted by a same amino acid or different amino acids;
(6) a reductase according to (3) or (5) above, wherein the amino acid at amino acid position 54 is substituted by a non-aromatic amino acid;
(7) a reductase according to (3) or (5) above, wherein the amino acid at amino acid 54 is substituted by glutamine, glycine, serine, threonine, cysteine, asparagine, alanine, valine, isoleucine, methionine, lysine, arginine, aspartic acid, glutamic acid, tyrosine, proline or histidine;
(8) a reductase according to (4) or (5) above, wherein the amino acid at amino acid position 104 is substituted by cysteine;
(9) a reductase according to (1), (2), (3), (4) or (5), wherein said further substitution comprises a substitution at amino acid position 245 or 271 or at both of the amino acid positions 245 and 271;
(10) a reductase according to (9), wherein said further substitution comprises a single amino acid substitution at amino acid position 245 in the amino acid sequence of SEQ ID NO:1;
(11) a reductase according to (9) above, wherein said further substitution comprises a single amino acid substitution at amino acid position 271 in the amino acid sequence of SEQ ID NO:1;
(12) a reductase according to (9) or (10) above, wherein the amino acid at amino acid position 245 is substituted by arginine;
(13) a reductase according to (9) or (11) above, wherein the amino acid at amino acid position 271 is substituted by aspartic acid;
(14) a reductase according to (1) above, wherein
(a) the amino acid at amino acid position 54 is substituted by glutamine and the amino acid at amino acid position 104 is substituted by cysteine;
(b) the amino acid at amino acid position 54 is substituted by glutamine,
the amino acid of the position 104 is substituted by cysteine and said further substitution comprises substitution of the amino acid at amino acid position 271 by aspartic acid;
(c) the amino acid at amino acid position 54 is substituted by glutamine and
the amino acid at amino acid position 104 is substituted by cysteine, and
said further substitution comprises
the amino acid substitution at amino acid position 245 by arginine, and
the amino acid substitution at amino acid position 271 by aspartic acid;
(d) the amino acid of the position 54 is substituted by glutamine, and said further substitution comprises the amino acid substitution at amino acid position 245 by arginine;
(e) the amino acid of the position 54 is substituted by glutamine, and
said further substitution comprises
substitution of the amino acid at amino acid position 245 by arginine, and
substitution of the amino acid at amino acid position 271 by aspartic acid; or
(f) the amino acid at amino acid position 54 is substituted by glutamine and said further substitution comprises substitution of the amino acid at amino acid position 271 by aspartic acid;
(15) a polynucleotide comprising a nucleotide sequence that encodes the amino acid sequence of the reductase according to (1) or (8) above;
(16) a vector comprising the polynucleotide according to (15) above;
(17) a transformant comprising the polynucleotide according to (15) above or the vector according to (16) above;
(18) a vector according to (16) above, which further comprises a polynucleotide having a nucleotide sequence that encodes the amino acid sequence of a protein capable of converting NADP (an oxidation-type β-nicotineamide adeninedinucleotide phosphate) or NAD (an oxidation-type β-nicotineamide adeninedinucleotide) into NADPH or NADH (reduction-type);
(19) a transformant according to (17) above, which further comprises a polynucleotide having a nucleotide sequence that encodes the amino acid sequence of a protein capable of converting NADP (an oxidation-type β-nicotineamide adeninedinucleotide phosphate) or NAD (an oxidation-type β-nicotineamide adeninedinucleotide) into NADPH or NADH (reduction-type).
(20) a production method for (S)4-halo-3-hydroxybutyrate ester, which comprises reacting 4-halo-3-oxobutyrate ester with the transformant according to (17) or (19) above or a treated material thereof;
(21) a method for modifying an enzyme, comprises substituting at least one single amino acid at amino acid positions 54 and 104 in the amino acid sequence of SEQ ID NO: 1, thereby selectivity of said enzyme is improved;
(22) a production method for a modified enzyme gene, which comprises replacing a codon that corresponds at least one of the amino acids at amino acid positions 54 and 104 of an amino acid sequence of SEQ ID NO: 1, with a codon that corresponds to the another amino acid(s), in a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1.